This invention relates to a process in purification and/or concentration of the factor VIII complex. It is started from a preparation prepared in a manner known per se, in which the factor VIII complex is enriched. The preparation is mostly in the form of a precipitate.
The factor VIII complex is lacking or its activity is reduced in hemophilia of type A and in v. Willebrand's disease. The symptoms in these diseases are often serious bleeding in joints and muscles and from mucous membranes.
The factor VIII complex takes part in the biochemical reactions promoting the coagulation of blood. In the coagulation of blood, an insoluble polymer, fibrin, is formed from a soluble protein, fibrinogen. The reason for the polymerization or formation of fibrin is an enzymatic change of the fibrinogen molecule, which is caused by the enzyme trombin (factor IIa). This enzyme is formed from protrombin (factor II) under the influence of an enzyme, called factor Xa. Said enzyme is also present as a zymogen in the blood before the coagulation. The zymogen form is called factor X. The conversion of factor X to factor Xa also takes place enzymatically by means of an enzyme called factor IXa. The factor VIII complex takes part in this reaction (as a co-factor) together with calcium and phospholipide. In the following scheme the most important reactions in the coagulation (clotting) of blood are illustrated. Totally a great number of various factors take part here. ##STR1##
The factor VIII complex consists of at least two components, one of which is called the factor VIII:C, in which C means that this component is responsible for the coagulation activity of the complex in the reaction chain shown. This component is considered to contain the antigen proved by means of antibodies, which are developed in certain persons suffering from hemophilia and which prevent the coagulation activity of factor VIII:C. The antigen is called F VIII:CAG. The other component has been called factor VIII-RAG or F VIII-related antigen. This antigen is different from the antigen F VIII:CAG. The factor VIII:C and the (antigen) factor VIII:CAG are lacking in hemophilia of type A in a serious form. In this disease there is a normal content of factor VIII:RAG. In v. Willebrand's disease, there is a lack of factor VIII:RAG in the blood and a corresponding lack of factor VIII:C. For persons suffering from a serious form of v. Willebrand's disease the lack of factor VIII:RAG is almost total and the content of factor VIII:C is about 5% of the normal content. In v. Willebrand's disease, the activity of the so-called factor VIII:RCF is highly reduced. This activity is an expression of a component in plasma which causes agglutination of trombocytes in the presence of the antibiotic "Ristocetin". It is now considered that the activity of the factor VIII:RCF is an expression of the component or components in plasma which has (have) the factor VIII-related antigen (factor VIII:RAG). The lack of factor VIII:RCF in v. Willebrand's disease has been found to be correlated to the prolonged capillary bleeding time existing in this disease and which is an expression of a defective trombocyte function. This function is normal in hemophilia type A, whereas the coagulation time of the blood is prolonged due to the reduced content of factor VIII:C. In v. Willebrand's disease, the coagulation time is also prolonged as there is also a lack of factor VIII:C in this disease, especially in serious forms thereof. The lack of factor VIII:C in v. Willebrand's disease is considered to be a consequence of the lack of factor VIII:RAG/RCF, which seems to act as carrier molecule for factor VIII:C. In accordance with the existing values, one can illustrate the factor VIII-complex schematically in the following way: EQU F VIII:RAG/RCF==F VIII:C/CAG
A number of processes are known at present for the preparation of plasma concentrates for clinical use, which contain the factor VIII complex or parts thereof. The complex can be precipitated from Cohn's fraction I with alcohol. Additional concentration of the complex can be carried out by extraction of inert protein whith about 1 M glycine solution in cold. Fractionation of plasma with ether or tannic acid has also been used for the preparation of factor VIII concentrates. Concentrates prepared by precipitation in cold of the factor VIII complex have been widely used due to the simplicity of the method. In certain cases, polyethylene glycol has been used for precipitation of the factor VIII complex. Variants of the glycine method, in which either other amino acids than glycine have been used or glycine has been used for precipitation of the factor VIII complex are also known.
Concentrates prepared according to the glycine method contain all factors in the factor VIII complex, but the specific activity is low, and therefore great volumes of solution must be injected in the treatment of hemophilia. Preparations with a high specific activity have often been found to lack the factor correcting the prolonged bleeding time in v. Willebrand's disease.
As to the yield of the coagulation active part (F VII:C), this is low in most preparation processes or between 20 and 30% at best.
In the treatment of hemophilia, it is of a great importance to use concentrates of the F VIII complex with a high specific activity of all the factors included in the complex. Hemophilia A as well as v. Willebrand's disease can be treated with such a concentrate. A specific activity which is 200-300 times greater than that in plasma also permits preparations containing the necessary therapeutic dose in a small volume (5-10 ml). This makes the treatment easier as the dose can be administered in an injection syringe. This also makes it possible to carry out the treatment for example at home by the sick person himself as is usual in the treatment of diabetes with insulin.
In most countries, the supply of blood is unsatisfactory in respect of the possibilities of preparing sufficient amounts of factor VIII complex for treatment of hemophilia from available blood volumes. This situation might be considerably improved if the percentage yield of the factor VIII complex could be increased in preparation from blood.